HIV-1 Tat protein within the central nervous system plays a pivotal role in the neurotoxicity and cognitive impairment evident in HIV-associated neurocognitive disorders (HAND). Converging lines of clinical observations, supported by imaging, neuropsychological performance, and postmortem examinations, have implicated that dysregulation of dopamine (DA) signaling is a risk determinant of HAND. Drugs of abuse, such as cocaine have been shown to exacerbate the severity of HAND by enhancing viral replication. Tat and cocaine synergistically increase synaptic DA levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Therefore, an intervention for HIV infection-induced dysfunction of DA system has the potential to improve neurocognitive function in patients with the early- stage of HAND. Currently, it is unclear how Tat influences the DA system in the brains of HIV- infected patients with HAND, thereby producing neurocognitive impairment. Our studies will identify the intermolecular interactions between Tat and human DAT (hDAT) and explore how Tat potentiates cocaine-induced inhibition of DA transport, leading to DA dysregulation for both mechanistic and interventional purposes. Recently, we identified key residues for hDAT interacting with Tat, which are critical for Tat-induced inhibitio of DAT uptake. For example, we found that DAT Tyrosine88 replaced by Phenylalanine (Y88F) displays normal surface DAT expression and DA uptake but attenuates Tat-induced inhibition of DA transport. Moreover, we found that Y88F partially attenuates the allosteric modulatory effects of SRI-20041, a novel DAT allosteric modulator, on DAT function. These studies led us to the novel hypothesis that Tat acts via the unique tyrosine88 site to perturb the DAT regulatory network that normally sustains concentrative DA transport, resulting in DA-linked neuropsychiatric dysfunction prominently featured in HAND. We will generate DAT Y88F knock-in mouse line harboring a doxycycline- inducible Tat transgene, and determine the impact of DAT Y88 on Tat-induced inhibition of DA transport and associated cognitive/behavioral deficits in Tat transgenic mice (Aim 1). Moreover, we will determine whether microinfusion of SRI-20041 into prefrontal cortex alleviates cognitive and behavioral deficits in Tat transgenic mice (Aim 2). This exploratory research will uncover critical functions of DAT important for Tat-induced cognitive/behavioral deficits and provide a novel mechanistic basis to identify targets on the DAT for developing compounds that specifically block Tat binding site(s) in hDAT, thereby stabilizing physiological DA transmission.